Tetracycline fermentation



Aug. 17, 1965 Original Filed Feb. 12, 1962 RE FLECTANCE J. A. GROWICH, JR., ETAL Re. 25,840

TE'IRACYCLINE FERMENTATION 8 Sheets-Sheet 1 REF LECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH AUREOFACIENS STRAIN T-IISS IN CORN STEEP MEDIUM WAVELENGTH (MILLIMICRONS) AR AT 420 mA 27 mvsmons AR AT 430 m I8 JOHN ANDREW GROWICH, JR NICHOLAS DEDUCK Aug. 17, 1965 Original Filed Feb. 12, 1962 REFLECTANCE J. A. GROWICH, JR.. ETAL 25,340

TETRACYCLINE FERMENTATION 8 Sheets-Sheet 2 F lG.-2 REFLECTANCE CURVE 0F WHOLE HARVEST MASH OBTAINED WITH AUREOFACIENS STRAIN T-l40 RN STEEP MEDIUM TOO WAVELENGTH (MILLIMICRONS) AR AT 420 m,u= 32 AR AT 430 mu I8 INVENTORS JOHN ANDREW GROWICH, JR.

ICHO AS D UCK B v 2'2 ATTO Y 8 Sheets-Sheet 3 Aug. 17, 1965 J. A. GROWICH, JR.. ETAL TETRACYCLINE FERMENTATION Original Filed Feb. 12, 1962 REFLECTANGE CURVE 0F WHOLE HARVEST MASH OBTAINED WITH i AUREOFACIENS STRAIN T IN CORN STEEP MEDIUM 5 5 4. 0 O 0 O 0 0 0 0 O 0 O 0 MOZFrOM EME m m 0 O WAVELENGTH (MILLIMICRONS) AR AT 420 m 59 mvENroRs AR AT 430 m -9 JOHN ANDREW GROWIGH,JR.

NICHOLAS 01-: van BY 4/, A ATTONEY Aug. 17, 1965 Original Filed Feb. 12, 1962 REFLECTANCE J. A. GROWICH, JR.. ETAL Re. 25,840

TETRACYGLINE FERMENTATION 8 Sheets$heet 4 F lG.-4 REF LECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITHi AUREOFACIENS STRAIN T-225 IN CORN STEEP MEDIUM WAVELENGTH (MILLIMICRONS) AR AT 420 mu 72 AR AT 430 III U. 3

INVENTORS JOHN ANDREW GROW|GH,JR.

NICHOLAS DUCK BY ,1

// I I I ATTOR EY 1955 J. A. GROWICH, JR., ETAL R 25,840

TETRACYGLINE FERME'NTATION 8 Sheets-Sheet 5 Original Filed Feb. 12, 1962 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH AUREOFACIENS STRAIN 8-2308 IN CO STEEP MEDIUM 0 O 0 O O O OOO QO O WAVELENGTH (MILLIMICRONS) um Y Ov E WWO EIU O w n A I ML wm w w 3 6 W S Mm o o 2 3 4 4 A m R R A A REFLECTANCE Aug. 17, 1965 J. A. GROWICH, JR.. ETAL 25,340

TETRAGYGLINE FERMENTATION Original Filed Feb. 12, 1962 8 Sheets-Sheet 6 F lG.-6 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH AUREOFAGIENS STRAIN 5-2589 IN CORN STEEP- MEDIUM 400 5 WAVELENGTH (MILLI MICRONS) AR AT 420 my. 94 INVENTORS AR AT 430 LM 36 JOHN ANDREW GROWICH,JR.

ICHOLS D UCK ,4

ATIQR EY Aug. 17, 1965 Original Filed Feb. 12, 1962 REFLECTANCE J. A. GROWICH, JR., ETAL 25,840

TETRACYCLINE FERMENTATION 8 Sheets-Sheet 7 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH AUREOFAGIENS STRAIN 8-2589 IN SYNTHETIC MEDIUM WAVELENGTH (MILLIMICRONS) AR AT 420 m ns AR AT 430 mm a7 INVENTORS NICHOLAS D UCK tg/I1 M I ATIOR JOHN ANDREW GROWICH,JR.

1955 J. A. GROWICH, JR, ETAL 25,340

TETRACYCLINE FERMENTATION Original Filed Feb. 12. 1962 8 eets-Sheet 8 FIGrB REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH iAUREOFACIENS STRAIN 8-2589 IN DRIED WHEY MEDIUM REFLECTANCE WAVELENGTH (MILLIMICRONS) AFI AT 420 (HAL I29 INVENTORS AR AT 430 m 66 JOHN ANDREW GROWICH,JR

NICHOLAS DED CK ATTOR EY United States Patent Olfice Re. 25,840 Reissuecl Aug. 17, 1965 25 840 TETRACYC LIN E FERMENTATION John Andrew Growich, In, New City, N.Y., and Nicholas Deduck, Rahway, N.J., assignors to American Cyana- Rn Iid Company, New York, N.Y., a corporation of tune Original No. 3,092,556, dated June 4, 1963, Ser. No. 172,394, Feb. 12, 1962. Application for reissue June 9, 1964, Ser. No. 384,817

Claims. (Cl. 19580) Matter enclosed in heavy brackets appears in the original patent but forms no part of this reissue specification; matter printed in italics indicates the additions made by reissue.

This invention relates to the production of tetracycline by fermentation and, more particularly, is concerned with certain novel mutant strains of Streptomyces aureofaciens which possess the property of producing tetracycline to the exclusion of chlortetracycline irrespective of the chloride ion content of the fermentation medium.

It has been known for some time that microorganisms of the species Streptomyces aureofaciens which produce tetracycline also simultaneously produce chlortetracycline. T production of chlortetracycline is objectionable when tetracycline is the principal product sought to be produced. Generally, while specification standards permit small quantities of chlortetracycline to be present in specification quality tetracycline, the presence of any sizable quantities of chlortetracycline is objectionable. Moreover, the presence of these two antibiotics in any sizable amounts in the fermentation mash involves difiicult problems of separation in the refining or extraction procedures. It is possible, of course, to extract the antibiotics from the fermentation mash and by selective refining procedures to elfect a separation of the antibiotics. However, the refining procedures for effecting separation of the antibiotics are not without some difiiculty and they will usually involve some loss in total antibiotic potency. Moreover, chlortetracycline, which in those instances where tetracycline is the principal product of the fermentation, maybe considered a contaminant and is customarily discarded since it is usually not present in sufficient quantity to warrant the expense of a separate purification procedure to bring it up to specification standards. Heretofore, chloride ion deprivation or the use of chlorination inhibitors have been means available for suppressing the formation of chlortetracycline.

The present invention is based upon the discovery that certain novel mutant strains of Streptomyces aureo-faciens produce tetracycline, to the total exclusion of chlortetracycline, by fermentation. These novel mutant strains of Streptomyces aureofaciens are, in essence, chloride ionignoring strains in that they produce tetracycline exclusively regardless of the concentration of chloride ion in the medium.

The novel mutant strains of the present invention are strains of the species S. aureofaciens. The S. aureofaciens strains described below [since they] are all direct descendents of the chlortetracycline-producing S. aureofaciens A-337 soil isolate described in US. Patent No. 2,482,055 to Duggar, and deposited at the Northern Regional Research Laboratories, Peoria, Illinois, and in- Strain: ATCC No. T-l35 13,908 T- 13,909 Tl43 13,910 T-225 A 13,911

In general, the novel mutant strains of the present invention are characteristic of the species S. aureof-aciens but differ from previously described strains of S. aureofaciens, not only in pigmentation, but also in the colors of the whole harvest mashes obtained therewith. The colors of the whole harvest mashes obtained by fermentation with the novel mutant strains of the present invention all exhibit a value of AR at 420 m which is greater than the value of AR at 430 m FIGURES 1 through 8 of the drawings are spectrophotometric reflectance curves of one-centimeter glass cells filled with the whole harvest mashes obtained with the novel mutant strains of S. aureofaciens of the present invention. The spectrophotometric reflectance curve of a material constitutes a permanent record that does not require the maintenance of a sample. Furthermore, the units in which the curve is expressed are understood and accepted in every civilized country. In FIGURES 1 through 8, the wavelength of light in millirnicrons is plotted as the abscissa against the reflectance as the ordinate. The wavelength of light has been adopted internationally as the fundamental standard of length to which all other standards of length are referred. T hcse spcctrophotometric reflectance curves were determined with a standard spectrophotometer using magnesium carbonate as a reference.

The letter R denotes the reflectance value at some particular wavelength, for example, R is the reflectance value at a wavelength of 500 mn. The symbol AR denotes the vertical distance betwcen the reflectance curve (when both percent reflectance and wavelength are plotted on linear scales) and a straight line drawn through the reflectance curve intercepts at 400 m and 550 m This graphical determination of the values of AR at 420 run and at 430 mu may be readily carried out and in every case the novel mutant strains of the present invention impart a color to the whole harvest mashes such that AR at 420 my is greater that AR at 430 Ill/L.

The mathematical determination of the values of AR at 420 my. and at 430 m may also be accomplished by means of the following equations:

AR at m/J.=20(R55 (R429) AR at 430 mu:30(R IZOQR 15001 vherein R R R and R are the reflectance values t wavelengths at 400 m 420 mg, 430 m and 550 mu, espectively. This mathematical determination is not delendcnt upon the scale used for either percent reflectance J1 wavelength.

A whole harvest mas is the untreated mash obtained ifter the fermentation has proceeded to the point where oiosynthesis of the primary product has stopped for all practical purposes. Generally, the antibiotic potency of the fermentation mash ceases to rise appreciably after the fermentation has proceeded .for from about 140 to about 160 hours.

To illustrate the visual color variations among the novel mutant strains of S. aureofaciens which produce tetracycline exclusively, these strains were grown on cornsteep agar and the following observations were made.

COLOR OBSERVA'TIONS: S. AUREOFACIENS: A1 4 CORN STEEP AGAR: SIX DAY INCUBATION AT 26.5" C.

Strain Single colonies Mass growth Dark luggage tan Maple.

Orange rust Do. Oak brown Light brown.

Yellow maple.

1 Colors according to the Color Harmony Manual, Third Edition, Container Corporation of America.

COLOR OBSERVATIONS: 1 8. AUREOFACIENS: APG CORN- STEEP AGAR: SIX DAY INCUBATION AT 26.5 C.

Strain Single colonies Mass growth Light spice brown. Maple.

Colors according to the Color Harmony Manual, Third Edition, Container Corporation of America.

FORMULATION OF AP4 AGAR FORMULATION OF APB AGAR Same as AP4 agar, except cornsteep level is 6 grams per liter.

The novel mutant strains of S. aureofaciens of the present invention which produce tetracycline exclusively possess the same general characteristics as do the strains which produce both chlortetracycline and tetracycline, and differ among themselves in the same general manner that the tetracycline-producing and chlortetracycline-producing strains difier from each other as has been described in a number of scientific papers which have been published, The data appearing below will serve to further distinguish the novel mutant strains of S, aureofaciens of the present invention from previously known strains of S. aureofaciens such as the original A-377 strain available as NRRL-2209.

The novel Streptomyces aureofaciens mutant strains were differentiated from S. aureofaciens A-377 (NRRL- 2209) by observation of growth characteristics on various media incubated at 26.5 C.

(1) GLYCEROL ASPARAGINE BEEF EXTRACT AGAR Streptomyces aureoiaciens Strain T-l35 Strain T-l40 Growth Good, hyaline; apigmentons to topaz.

Good, hyalinc; aplgmentons to topaz.

Aerial hyphae.. Slight becoming moder- Slight becoming moderate, white. ate; white. Sporulation- None None. Ditiusible pigdo Do.

ment. Reverse Hyaline; topza becoming Hyalino; topaz becoming oak brown. oak brown.

Strain T443 Strain T-225 Growth Good, hyaline; dark lug- Excellent.

gage an. Aerial hyphae... Sparse to fair; white be- Abundant to profuse;

coming silver grey. white becoming light fawn 1 to beaver. Spornlatlon. Fair .c Profuse. Difiusibie pig- Light amber Yellow.

merit. Reverse Hyaline; deep brown Dark brown mahogany.

Strain A-377 Growth... Good Aerial hyphee Slight to fair; white to light gray. Stimulation.-- Fair Difiusable pig- Light yellow ment. Reverse Yellow to light orangeyellow.

Colors according to the Color Harmony Manual, Third Edition, Container Corporation of America.

(2) DEXTRIN CZAPEK-DOX AGAR percent Bacto agar..

Distilled wa I Post sterilization pH 7. 2

Streptomyoes aureolaciens Strain T- Strain T- Growth--- Slight, thin, semitrans- Slight, thin, semitranslucent; white. lucent; white. Aerial hyphae-.- Slight; white None. Sporulation None Do. Dittusible pigdo Do.

ment. Reverse Whit/e, translucent a. White, translucent.

Strain T-l43 Strain T-225 Growth Slight, thin semitranslu- Slight, thin, semi-opaque cent; white. w to. Aerial hyphae... Slight; white Slight, white. Sporulation. None None. Diffusible pigdo D0.

ment. Reverse White; translucent White; translucent.

Strain A-377 Growth Good Aerial hyphue. Abundant, mouse gray 1 to lead grey; waterwhite surface globules. Sporulation. Profuse Dlfiusible pig- Trace; pale yellow merit. Reverse Apigmentous; pink trace 1 Colors according to the Color Harmon Man oil I i l d Container Corporation of America. y l T I:

(3) AP4 CORNSTEEP AGAR (5) Q4 CORNSTEEP AGAR Cornsteep "percent. 4 Cornsteep .-grams 9 Sucrose Sucrose. MgSO4.7HzO.... MgSO -7H2O.. KHflPOi .do.... (NHQ HPO (NI-IMHPO; (1 2 Crude agar.. gg g g gg -a Water, q.s.-. Post ....r1r..aaa1;ii;:: -.:d 62 5 Wham PH Streptomyces aureofacines 1O Streptomyces aureofacicns Strain T-135 Strain T-MD Strain TD-135 Strain T-140 Growth Moderate Moderate. Growth Abundant Profuse. Aerie] hypha.e Scarce becoming abun- Moderate; beaver 1 to I Aerial hyphae... Abundant; fawn l to Profuse; fawn 1 to dent; beaver. chocolate. beaver. beaver. Sporulation.-- Moderate to abundant. Moderate. Sporulation Abundant Abundant. Soluble pigment. Orange-brown Orange-brown. Soluble pigment. Deep orange-brown Deep orange-brown. Reverse Light brown to chocolate Chestnut brown to deep Reverse Copper brown to deep Copper brown to deep brown. brown. brown. brown.

Strain T-143 Strain 'I225 Strain T-143 Strain T-225 Growth Abundant Abu dant. Growth Profuse Profuse. Aerial hyphae... Abigidant; fawn to bea- Profuse; chocola e- Aerial hyphae... Profuse; beige 1 to mist Profuse; chocolate.

v brown. Sporulation Moderate to abundant. Prof se. S orulation- Profuse Profuse. solllblfi D E Light Orange-brown Light (Range-brown- Soluble pigment. Deep orangebrown... Deep umber. Rev s D brOWIl to dark Dark 1059 brown to Reverse Deep brown to chocolate Ebony brown.

brown. ebony br w brown.

Strain A-377 Strain A377 a Growth Excellent Growth Excellent; pale yellow Ae hyp n Abundant; fawn Aerial hyphae... Profuse; dark brown. Sporulation- Profuse Sp0rulati0i1. Profuse Solu me t- Light y w to amber- Soluble pigment. Orange-brown Reverse Llght tan Reverse Orange to orange-yellow.

1 Colors according to the Color Harmony Manual, Third Edition, Container Corporation of America, 1 Colors according to the Color Harmony Manual, Third Edition,

Container Corporation of America.

(4) A1 6 CORNSTEEP A GAR Cornsteep.- Sucrose-. MgSOMHiO. K IP04 (NH-I)IHPO| aeto agar. Tap water, q.s Post sterilization pH Streptomyces aureofaciens Strain T-135 Strain T440 Growth Abundant Abundant. Aerial hyphae.-- Moderate to abundant; Moderate to abundant;

fawn l to mist brown. beaver 1 to fawn. Bporulation- Moderate to abundant. Profuse. Soluble pigment. Orange to orange-brown..- Orange to orange-brown. Reverse Chestnut brown to deep Chmstnut brown to deep brown. brown.

Strain T-lAB Strain T-225 Growth Abundant Abundant. Aerial hyphae--- Slight to moderate; beige Profuse; chocolate.

to rnist brown. Sporulation- Fair Profuse. Soluble pigment. Orange-brown Ora nge-brown. Reverse Chocolate brown Ebony brown.

Strain A-377 Growth Excellent Aerial hyphae.-- Moderate to abundant;

awn. Sporulation Profuse Soluble plgme Orange-brown. Reverse.- Orange to orange-yellow.

Manual, Third Edition,

(6) OTHER AGAR MEDIA Medium Streptomyoes aureofaciens Nutrient again.

Glucose Asparagine Meat extract Agar.

Waksmans agar.

Purple milk"-..

Potato slants-.

Reverse: white to polo yellow.

Good growth; hyaline,

apigmentous to pale orange. N o aerial hyphae. Reverse: apigmentous to pale orange.

Good growth, semiopaque; white to pale orange. Aerial hyphrte: sparse becoming abundant; white to fawn. Sporulatlon: spars becoming abundant. Reverse: dark luggage ten to dark spice brown. Light amber soluble pigment.

Pale yellow 1 growth collar. Partial peptonizaF tlon, pH 7.1.

Profuse, moist. nodulated growth; chestnut brown 1 to dark spice brown. Rust brown soluble pigment.

Strain T435 Strain T-MO Poor to fair growth; Poor growth; white to white to pale yellow. 1 pale yellow. N o aerial No aerial hyphae. hyphae. Reverse:

white to pale yellow.

Good growth; hyaline,

apignientous to pale orange. Slight aerial hypliue formation; white. Reverse: upigmentous to pale orange.

Good growth, semiopaquc; white to pale orange 1 becoming orange with age. Aerial hyphae: sparse becoming abundant; white to beaver. Sporulation: sparse becoin profuse. Reverse: eige to copper brown. Trace of light amber soluble pigment.

Pale yellow growth collar. Partial poptonization, pH 7.0.

Profuse moist, nodulated growth; light spice brown. Barn red soluble pigment. Slight white aerial liyphae formation.

1 Colors according to the Co tainer Corporation of America.

Streptomyces aureofaeiens Medium Strain T443 Strain T-225 Nutrient agar Ioor growth: white to Poor to fair owth: white light orange-yellow. to golden rown No No aerial hyphae. aerial hyphae. Re- Rcverse: white to light verse: translucent, orange-yellow. white to adobe brown.

No soluble pigment. Glusoce aspara- Good growth: hyaline. Abundant rowth. Progine meat expale yellow to orange. fuse aeria hyphae; tract agar. Aerial mghae: slight, beaver. Sporuletion:

white. averse: hyprofuse. Reverse: dark aline; pale yellow to eggplant. Very light copper brown to deep amber soluble pigment. brown Waksmans Good growth: light tan Abundant growth.

agar. to topaz. Aerial hy- Abundant aerial hyghae: fair to abundant, phae; taupe brown I to eige brown to town. chocolate. Reverse: Sporulation: poor beebony. Light amber coming proiuse. Resoluble pigment. verse: dark luggage ten to deep brown. Lig t amber to amber soluble pigment. Potato slants Profuse, moist, nodu- Profuse, moist, smooth lated growth; beaver l nodulated growth; to light spice brown. orange rust 1 to oak Dense white aerial hybrown becoming choeo hae at isolated foci. late with age. Orange ust brown soluble brown soluble pigment. pigment. Purple milk.. Pale yellow 1 growth col- Heavy pale yellow 1 lar. Little significant pH change or apparent peptonization, pH 0.7.

growth collar. Partial peptonlzation, pH 6.78. No soluble pigment.

Colors according to the Color Harmony Manual, Third Edition Container Corporation of America.

Streptomyees aureofa/ciens Medium Strain A4577 Glucose asparagine meat extract agar.

Waksman's agar.-."

Potato slants Purple milk Colors according to the Color Harmony Manual, Third Edition Container Corporation of America.

(7) MICROSCOPIC OBSERVATIONS Streptomyces aureofaeiens Medium Strain T-l35 Strain 'I140 Mycelium Spores Mycelium Spores AP4 corn- Flexuous, spheroidal Flexuous, spheroidal steep agar. continuto ovoidal. continuto ovoidal. ous Diam. 0.8 ous, Diam. 0.8 branched. to 1.5 branched. to 1.5 Diam. 0.8 Diani. 0.8 to 1.0;; to 1.0 n Q4 agar Flexuous, spheroidal Flexuous, Spheroidal continuto ovoidal. entlnu to ovoidal ous. Diam. 1.0 ous, Diam. 1.0 branched. to 1.2;. branched. to 1.2;. Diem. 0.8 Diam. 0.8 130 1.0 to 1.0 Waksmans Flexuous, spheroidal Flexuous, spheroidal agar. continuto ovoidal. continuto ovoldal ous, Diani. 1.2 ous, lliain. 1.0 branched. to 1.5;. branched. to LZ Diem. 0.3 lJiam. 0.8 to 1.0,.. to 1.0,.

Streptomyees aurooiaciens Medium Strain T-l43 Strain 13-225 Mycelium Spores Mycelium Spores Glycerol Flexuous, spheroidal Flexuous, spheroidal asparagine continuto ovoidal. continuto ovoidal. meat ous, Dium. 1.0 on Diam. 1.2 extract branched. to 1.2 branched. to 1.5 agar. Diana. 0.8 Diain. 0.8

to 1.0 $0 1.0 AP4 corn- Flcxuous, spheroidal Flexuous, Spheroidal steep agar. continuto ovoidal eontinuto ovoidal. ous, Diam. 0.8 ous, Diain. 1.0 branched. to 1.5;. branched. to 1.2 Diam. 0.8 Dialn. 0.8 t0 1.2;. 1.0 4.

Streptomyces aureofaciens Medium Strain A-377 Mycclium Spores Glycerol asparagine Flexuous, continuous, spheroidal to ovoidal.

branched. Diem. 0.8 Diam. 1.0 to 1.5

spheroidal to ovoidal. Diam. 1.2 to 1.5;.

meat extract agar.

AP4 cornsteep agaL. Floxuous, continuous,

branched. Diem. 1.0 60 1.214.

the fermentation are generally the same as for the presently known methods of producing chlortetracycline by fermentation. That is, the fermentation medium contains the usual nutrients and minerals substances. Suitable nutrient substances include starch, dextrose, cane sugar, glucose, molasses, soybean meal, milk solids, yeast, meat extracts, peptonc, urea, cornsteep liquor, distillers solubles, fish meal and other conventional substances. The inorganic salts include such things as calcium carbonate, ammonium sulfate, ammonium chloride, and salts of the various trace elements such as manganese, cobalt, zinc, copper, iron and the like.

The other general conditions of the fermentation such as hydrogen ion concentration, temperature, time, rate of aeration, preparation of the inoculum, sterilization inoculation and the like are conventional and may be similar to those for the production of chlortetracyclinc shown in US. Patent No. 2,482,055 to Duggar.

The recovery of the tetracycline from the fermentation liquor is conventional and need not be described as numerous methods for recovering tetracycline from fermentation liquors have already been published.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 Inoculum preparation of S. aurcofaciens strain T-l35 The conditions of Spores of S. aureofaciens strain Tl35 were washed from a streaked agar slant with sterile distilled water to form a suspension containing 60-80X10 spores/ml. A 0.33-ml. portion of this suspension was used to inoculate an eight-inch shaker tube containing 8 ml. of medium prepared according to the following formulation:

Sucrose "grams" 30 Ammonium sulfate do 2 Calcium carbonate do 7 Cornstcep ml 16.5 Tap water, q.s ml 1,000

novel mutant strains of the present invention are prepared in an identical fashion.

EXAMPLE 2 Chloramine-T test for tetracycline-chlortetracycline diiferentiation 1,000 ml. of distilled water). test, 0.5 ml. of the fermentation mash to be tested is mixed in a test tube with 20 ml. of working solution B. The tube is shaken intermittently for a period varying from 30 to 60 minutes. At the termination of this period of shaking, the test tube is examined for color. Mashes containing high concentrations of tetracycline tum wine red (a positive reaction), while mashes containing high concentrations of chlortetracycline develop a deep amber color. This Chloramine-T test is a further means of differentiating the novel mutant strains of the present invention from previously known strains of S. aureofaciens.

EXAMPLE 3 filtrate thus obtained is re-filtered as often as necessary to obtain a clear filtrate. A Z-ml. portion of 0.01 M AgNO is next added to a 2-ml. aliquot of the clear filtrate in a test tube.

presence of free unbound chloride ion in the whole mash samples.

EXAMPLE 4 Tetracycline production in shaker flask fennentations A. FULL CHLORIDE MEDIUM A full chloride fermentation medium for tetracycline production was prepared as follows:

(NH SO grams 5 CaCO do 7-9 NH CL do 1.5 FeSo -7H O do 0.4-0.6 MnSO '4H 0 do 0.05 ZnSO -7H O do 0.1 Cornsteep liquor do 20-30 Cottonseed meal do 0-2 Starch do 55 Lard oil ml 20 Tap Water, q.s ml 1,000

B. CHLORIDE-LIMITED MEDI UM A chloride-limited fermentation medium for tetracyline production was prepared as follows:

250-ml. Erlenmeyer flasks, sealed in each case with a cotton plug, and sterilized in an autoclave for 20 minutes at a pressure of pounds per square inch. After sterilization, each pair of -ml. aliquots were variously inoculat ed with 1.0 ml. of inocula prepared as in Example 1. The fermentation was carried out at 28" C. from 0 to 18 hours and then at 24 C. from 18 to 142 hours, at pH 6-7, on a rotary shaker operating at 180 revolutions per minute.

TABLE I Assays Chlora- Silver Strain N0. Medium mine-T nitrate Chlortetre- Tetratest I test eycline, cycline meg/ml. meg. /1ni.

T-l A I 2, 460 B 50 2, 510 T440 A 50 2, 210 B 50 2, 200 T443 A 50 3, 820 B 50 3, 470 T'225 A 50 3, 800 B 50 3, 990 8-2308 A. 50 6, 580 B 50 6, 520

l Chloramine-T test as per Example 2.

5 Silver nitrate test as per Example 3. D

3 The chlortetracyline assay method is not sensitive to less than 50 meg/ml. However, the cultures showed no ehlortetraeycline by paper t hr at graphy. om 0 EXAMPLE 5 Tetracycline production in shaker flask fermentationseffect of ammonium chloride concentration Fermentation medium A of Example 4 was prepared with levels of 0.0 0.2, 0.5, and 1.5 g./i. of NH Cl and a constant 5.0 g./l. level of (NI-1.9 Fermentation was carried out as described in Example 4, with the 1 Silver nitrate test; as per Example 3. 2 The chlortetracycline assay method is not sensitive to less than 50 meg/ml. However, the cultures showed no chlortetracycline by paper strip chromatography.

EXAMPLE 6 Response of tetracycline-producting starins to ultraviolet light The novel mutant strains of the present invention may be readily diflerentiated from previously known chlortetracycline-producing from established strains producing primarily other known tetracyclines such as 6-dimethyltetracycline, 7-chloro-6- dimethyltetracyciine, and 7-chloro-5a(1 1a)-dehydrotetracycline) by comparison of the fluorescence produced agar, prepared as follows:

do Tap water, q.s ml 1000 The pH of this medium was adjusted to 6.6 by means of a 50% solution of potassium hydroxide, then 17.5-ml. aliquots of the medium were dispensed into 25 x 150 mm. glass culture tubes and the tubes closed with cotton plugs. The culture tubes containing the medium were sterilized at 120 C. for 20 minutes, allowed to solidify in slanted position to provide maximum surface area, and refrigerated until needed. Spores of S. aureofaciens strain A-377 were washed from an agar slant with sterile distilled water to form a suspension containing 60-80 10' spores/ml. A 0.1 ml. portion of this suspension was used to inoculate a portion of the Q sporulation agar slants. After inoculation, the Q agar slants were incubated at 26.5-27.5 C. Sporulation was generally complete after 14 days. The same procedure was followed in the case of all the other microorganisms tested which are listed in Table III below.

When observed under ultraviolet light (3660 A.) during the -4 days incubation period, vegatative growth of tetracycline-producing strains became brilliant goldenorange while the butt became increasingly brilliant orange-yellow to yellow. During this phase of growth, chlortetracycline-producing strains may also exhibit a slight amount of fluorescence of a similar type. Simultaneously, the upper portion of the agar slant, where the medium thickness is minimal, fluoresces green-yellow for tetracycline-producing strains and -golden-yellow for chlortetracycline-producing strains. Further, as the slants mature, the tetracycline-producing strains retain the yellow fluorescent response in the agar butt to ultraviolet light at 3660 A. whereas the chlortetracycline-producing strains become pale blue.

A fermentation medium for tetracycline production was prepared according to the following formulation:

(NI-140 50 ..grams 5.58 CaCO do 7-10 NH Cl do 1.66 MHSO (70% tech. grade) do 0.08 Cornsteep liquor do 20-30 Starch do 47.5 Corn flour do 14.5 Lard oil ml 20-32 Tap water, q.s ml 1000 The fermentation procedures were carried out according to the directions given in Example 4, using the microorganisms listed in Table III below, with the results set forth in Table III below.

TABLE I11 Assays S. aureoieeiens Q agar slant fluores- Staln No. Chlortet- Tetracyeence at 3660 A recycline cline meg/ml. meg/ml.

50 0 Pale blue. 1 50 620 Brilliant yellow.

50 730 Do. 50 5, 670 Do. 50 4,140 Do. 50 6, 640 Do. 50 8, 770 Do.

1 The chlortetraeyline assay method is not sensitive to less than 50 meg/ml. However, the cultures showed no chlortetracycline by paper strip cinematography.

EXAMPLE 7 Characterization of tetracycline-producing strains of S. aureofaciens by spectrophotometric reflectance of the whole harvest mashes in cornsteep medium pie 1. Subsequent preparation of inocula of the novel mutant strains of S. aureofaciens employed followed the method of Example 1.

A fermentation medium of the following composition was prepared:

(NHQ SQ; grams 5.5-7.0 CaCO do 7-10 NH Cl do 1 5-2.0 MnSO (70% tech. grade) do 0.08-0.15 Cornsteep liquor do 20-30 Cottonseed meal do 0-5 Starch do 45-55 Corn flour do 14.5 Lard oil ml 20-35 Tap water, q.s ml 1000 After sterilization of thi medium in an autoclave for 20 minutes at a pressure of 15 pounds per square inch, 25 ml. aliquots in 250-ml. Erlenmeyer flasks were inoculated with 1.0 ml. portions of the inoculum prepared as described above. The fermentation was carried out at 28 C. from 0 to 18 hours and then at 24 C. from 18 to 142 hours, on a rotary shaker operating at 180 revolutions per minute. The same procedure was followed in the case of the other microorganisms employed which are listed in Table IV below.

Assays for tetracycline were performed according to the standard Hiscox method while chlortetracycline assays were obtained by a fluorimetric technique. The results are set forth in Table IV below. Reflectance curves were obtained for the whole harvest mashes by the use of a General Electric Co. type spectrophotometer, and are reproduced in FIGURES 1 through 6 of the drawings. The samples were run in a one-centimeter glass cell placed in the reflectance position at the back of the sphere; magnesium carbonate was used as the reference. The specular reflectance of the cell was included and a range of 400 mg to 700mg was scanned. The values of AR at 420 m and at 430 mg were determined and are set forth in table IV below.

TABLE IV Assays AR at S. aureotaciens Stain No. Chlortetra- Tetracyeycline, eline, er- 420 m 430 m percent by cent y weight weight The cultures showed no chlortetracyeline by paper strip chromatography.

"Ihe scales of reflectance and wavelength in Figures 1 through 8 are not linear with the (Eltllltities plotted. This is due to the particular plotting mec anism of the spectrophotometer used and in no way effects the calculation of the AR values.

EXAMPLE 8 Characterization of a tetracycline-producing strain of S. aureofaciens by spectrophotometric reflectance of the whole harvest mash in synthetic medium Spores of S. aureofaciens strain 8-2589 were washed from an agar slant with sterile distilled water to form a suspension containing 60-80Xl0, spores per ml. A

0.33 ml. portion of this suspension was used to inoculate an eight-inch shaker tube containing 8 ml. of a medium prepared according to the formulation disclosed in Ex ample 1.

A fermentation medium of the following composition was prepared:

CaCO "grams" 10.0 (NHQ)2SO4 d0 NH Cl do 1.5

13 Cornstarch grams 55.0 MgCl -6H O do 20 FeS -7H O do 0.06 ZnSO -7H O do 0.10 MHSO4'H2O d0 COCl '6H O d0 0.005 KCl dO 1.28 H PO do 0.36 L-methionine do 0.60 L-histidine do 0.80 Distilled H O, q.s ml 1,000

To this medium was added a lard oil-mineral oil combination, consisting of 3 parts of lard oil to 1 part of U.S.P. grade mineral oil, at the rate of 0.63 ml. per 25 ml. of medium. After sterilization of this medium in an auto clave for 20 minutes at a pressure of 15 pounds per square inch, 25 ml. aliquots in 250-ml. Erlenmeyer flasks were inoculated with 1.0 ml. portions of the inoculum prepared as described above. The fermentation was carried out at 28 C. from 0 to 18 hours and then at 24 C. from 18 to 142 hours, on a rotary shaker operating at 180 revolutions per minute.

The whole harvest mash assayed 8060 micrograms of tetraycyline per ml. according to the standard Hiscox assay method whereas the culture showed no chlortetracycline by paper strip chromatography. A reflectance curve was obtained for the whole harvest mash by the use of a General Electric Co. type spectrophotometer, and is reproduced in FIGURE 7 of the drawings. The sample was run in a one-centimeter glass cell placed in the reflectance position at the back of the sphere; magnesium carbonate was used as the reference. The specular reflectance of the cell was included and a range of 400 m to 700 m was scanned. The value of AR at 420 m was 118 whereas the value of AR at 430 m was 87.

EXAMPLE 9 Characterization of a tetracycline-producing strain of S. aureofaciens by spectrophotometric reflectance of the whole harvest mash in dried whey medium Spores of S. aureofaciens strain 8-2589 were washed from an agar slant with sterile distilled water to form a suspension containing 6080 10 spores per ml. A 0.33 ml. portion of this suspension was used to inoculate an eight-inch shaker tube containing 8 ml. of a medium prepared according to the formulation disclosed in Example 1.

A fermentation medium of the following composition was prepared:

Dried whey -grams Cornflour do Cornstarch do CaCo do (NH4)2SO4 dO. NH Cl do 2.0 M1150 (70% technical grade) do 0.140 CoCl -6H O do 0.005 Lactic acid do 0.650 Tap water, qs ml 1,000 To this medium was added 0.75 ml. of lard oil per 25 ml. of medium. After sterilization of this medium in an autoclave for 20 minutes at a pressure of pounds per square inch, 25 ml. aliquots in 250-ml. Erlenmeyer flasks were inoculated with 1.0 ml. portions of the inoculum prepared as described above. The fermentation was carried out at 28 C. from 0 to 18 hours and then at 24 C. from 18 to 142 hours, on a rotary shaker operating at 180 revolutions per minute.

The whole harvest mash assayed 6070 micrograms of tetracycline per m1. according to the standard Hiscox assay method Whereas the culture showed no chlortetracycline by paper strip chromatography. A reflectance curve was obtained for the whole harvest mash by the use of a General Electric Co. type spectrophotometer, and is reproduced in FIGURE 8 of the drawings. The sample was run in a one-centimeter glass cell placed in the reflectance position at the back of the sphere; magnesium carbonate was used as the reference. The specular reflectance of the cell was included and a range of 400 m to 700 mu was scanned. The value of AR at 420 mu was 129 whereas the value of AR at 430 m was 66.

This application is a continuation-in-part of our copending application Serial No. 51,974, filed August 25, 1960, now abandoned.

What is claimed is:

1. The process of producing tetracycline which comprises cultivating a strain of Streptomyces aureofaciens which produces tetracycline exclusively under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts until substantial quantities of tetracycline are produced, said tetracycline-producing strain of Streptomyces aureofaciens being [also] characterized by its ability to impart to the whole harvest mash a color characterized by a reflectance curve, when plotted linearly, wherein the vertical distance between the reflectance curve and a straight line drawn through the reflectance curve intercepts at 400 m and 550 lTl/L is greater at 420 mu than at 430 mu [-1, and by the failure of a mature agar slam culture thereof, obtained by the technique described in Example 6, to give a blue fluorescent response to ultraviolet light at 3660 A 2. The process of producing tetracycline which comprises cultivating Streptomyces aureofaciens ATCC No. 13908 under submerged aerobic conditions in an aqueous nutrient medium containing .assimilable sources of carbohydrate, nitrogen, and inorganic salts until substantial quantities of tetracycline are produced.

3. The process of producing tetracycline which comprises cultivating Streptomyces aureofaciens ATCC No. 13909 under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts until substantial quantities of tetracycline are produced.

4. The process of producing tetracycline which comprises cultivating Streptomyces aureofaciens ATCC No. 13910 under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts until substantial quantities of tetracycline are produced.

5. The process of producing tetracycline which comprises cultivating Streptomyces aureofaciens ATCC No. 13911 under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts until substantial quantities of tetracycline are produced.

References Cited by the Examiner The following referneces, of record in the patented file patent.

A. LOUIS MONACELL, Primary Examiner. 

